Modern silviculture often requires the planting of large numbers of genetically identical plants that have been selected to have advantageous properties. Production of new plants by sexual reproduction, which yields botanic seeds, is usually not feasible. Asexual propagation, via the culturing of somatic or zygotic embryos, has been shown for some species to yield large numbers of genetically identical embryos, each having the capacity to develop into a normal plant.
Somatic cloning is the process of creating genetically identical plants from plant tissue other than male and female gametes. In one approach to somatic cloning, plant tissue is cultured in an initiation medium that includes hormones, such as auxins and/or cytokinins, to initiate formation of embryogenic tissue, such as embryogenic suspensor masses, that are capable of developing into somatic embryos. The embryogenic tissue is then further cultured in a multiplication medium that promotes multiplication and mass production of the embryogenic tissue. The embryogenic tissue is then cultured in a development medium that promotes development and maturation of cotyledonary somatic embryos that may, for example, be placed on germination medium to produce germinants, and subsequently transferred to soil for further growth, or alternatively, placed within manufactured seeds and sown in soil where they germinate to yield seedlings. Manufactured seeds are described, for example, in U.S. Pat. Nos. 5,564,224; 5,687,504; 5,701,699; and 6,119,395.
The typical somatic embryogenesis process is laborious and inefficient. For example, one of the more labor intensive and subjective steps in the embryogenesis process is the selective harvesting of individual embryos suitable for germination. At the end of the development period, the embryos may be present in a number of stages of maturity and development. Those that are most likely to successfully germinate into normal plants are preferentially selected using a number of visually evaluated screening criteria. Typically, a skilled technician evaluates the morphological features of each embryo, such as the embryo's size, shape (e.g., axial symmetry), cotyledon development, surface texture, color, and the like, and manually plucks desirable embryos with a pair of tweezers and transfers the selected embryos to germination medium. The selection process is highly subjective, and the transferring of embryos to germination medium by hand remains a tedious, laborious, and ergonomically challenging process. Further, it poses a major production bottleneck when the ultimate desired output can be in the thousands of plants.
Efforts have been made to use instrumental image analysis for embryo selection to supplement or replace the visual evaluation performed by technicians. For example, an elaborate and complex classification method is disclosed in U.S. Publication No. 2007/0269096, which describes the classification of plant embryos by the application of classification algorithms to digitized images of plant embryos, and absorption, transmittance, or reflectance spectra of the embryos, to determine which embryos are likely to develop into germinants. Similarly, U.S. Pat. No. 7,610,155 describes using image and spectral data from known quality embryos to develop a classification model, using a classification algorithm, such as logistic regression (LR) analysis, to classify embryos as (i) embryos that likely will not germinate; (ii) embryos that may germinate with extra care; and (iii) embryos that will germinate with minimal care. The classification model is then applied to image and/or spectral data acquired from a plant embryo of unknown quality to determine the likelihood the embryo will develop into a germinant. Although determining the germination potential of embryos by classification modeling is a more objective process than selection of embryos by technicians, such methods involve the use of expensive instrumentation to collect the required images and data on each embryo, as well as extensive studies of embryos of known quality to develop the modeling system.
Thus, there exists a need for methods of selecting embryos that are most likely to successfully germinate into normal plants that simplify the process, reduce the need for selection by technician, and/or the use of expensive instrumentation, and increase the production rate to achieve commercial scale.
The present invention is directed to methods of sorting plant somatic embryos according to germination potential.